Assesment of metal inhibition of antioxidant enzyme glutathione reductase from rainbow trout liver

Abstract

Effects of Co⁺², Zn⁺², Ca⁺², Fe⁺², Mn⁺², Cr⁺³, Sn⁺² and Mg⁺² exposure of rainbow trout liver on the enzymatic activity of glutathione reductase (GR) were investigated. GR was purified from rainbow trout liver 1419-fold with a yield of 38.41% and a specific activity of 22.846 U/mg protein. Following enzyme isolation, inhibitory effects of Co⁺², Zn⁺², Ca⁺², Fe⁺², Mn⁺², Cr⁺³ and Sn⁺² were analysed. IC₅₀ values of the metal ions ranged from 42.2 to 657 µM, and the K_i constants ranged from 24.2 to 567µ. Co⁺², Zn⁺², Fe⁺² and Cr⁺³ exhibited competitive inhibition whereas others inhibited the enzyme in non-competitive manner. Cobalt was the most powerful inhibitor among others having the lowest IC₅₀ and K_i values. Magnesium exhibited activatory effect on the enzyme.

Keywords::

• Glutathione reductase
• rainbow trout
• inhibitor
• metal ion

Introduction

The undesirable biologic effects of oxidative agents such as free radical and reactive oxygen species are eliminated by enzymatic and non-enzymatic antioxidant defence systems. Enzymatic defence is provided by many enzyme systems, such as glutathione reductase, glutathione peroxidase, glutathione S-transferase, superoxide dismutase, catalase and DNA repair enzymes. Particularly, glutathione reductase (Glutathione: NADP⁺ oxidoreductase, EC 1.8.1.7; GR) is the key enzyme in the glutathione metabolism. Glutathione reductase is essential for the maintenance of cellular glutathione in its reduced form, which is highly nucleophilic for many reactive electrophils^1,2. This flavin enzyme is essential for reduction of glutathione disulfide (GSSG) to the reduced form (GSH), necessary for protection of the cells against oxidative stress as an antioxidant. GR catalyzes the reduction of glutathione disulfide (GSSG) at the expense of NADPH:

GSH is also a reaction partner for the detoxification of xenobiotics, it is a cofactor in isomerization reactions, and is a storage and transport form of cysteine^3,4. It maintains the thiol redox potential in cells keeping sulfhydryl groups of intracellular proteins in the reduced form⁵. Decreased GSH levels have been reported in several diseases, such as acquired immune deficiency syndrome (AIDS⁶), adult respiratory distress syndrome,⁷ Parkinson’s disease,⁸ and diabetes⁹. In addition, recent results suggest that GSH is essential for cell proliferation,¹⁰ and it plays a role in the regulation of apoptosis.¹¹ Alternatively, high GSSG concentrations inhibit a number of important enzyme systems including protein synthesis¹².

Metals are notable for their wide environmental dispersion from such activity; their tendency to accumulate in selected tissues of the human body; and their overall potential to be toxic even at relatively minor levels of exposure. Some metals, such as copper and iron are essential for life and play irreplaceable roles in, for example, the functioning of critical enzyme systems. Other metals are xenobiotics, i.e. they have no useful role in human physiology (and most other living organisms) and, even worse, as in the case of lead and mercury, may be toxic even at trace levels of exposure¹³. Exposure to heavy metals is an important problem of environmental toxicology. Most heavy metals are toxic to humans, animals and plants. Man is at great risk of suffering from health hazards associated with toxic metals because of bioaccumulation¹⁴.

GR has been purified from different tissue cells, using different purification procedures. All reported purification procedures involve several chromatographic steps^2,15. Effects of many drugs, metal ions and chemicals on human and fish GR enzyme activities have been investigated so far^2,16–19. However, no reports could be found in the literature on the effects of these metals on fish liver GR. Therefore, in the present study, we purified GR from rainbow trout and examined in vitro inhibition effects of some metal ions on the enzyme.

Materials and methods

Chemicals

Sephadex G-200, NADPH, GSSG, protein assay reagents and chemicals for electrophoresis were obtained from Sigma Chem. Co., Germany. 2′, 5′-ADP Sepharose-4B was obtained from GE Healthcare, Munich, Germany. All other chemicals used were of analytical grade and obtained from either Sigma-Aldrich or Merck, Germany.

Glutathione reductase enzyme activity

Glutathione reductase enzyme activity was measured by Beutler’s method²⁰. One enzyme unit is defined as the oxidation of 1 µM NADPH per min under the assay condition (25°C, pH: 8.0).

Preparation of the homogenate

Rainbow trout (200 ± 20 g) were obtained from the Department of Fishery Sciences, Agriculture Faculty at Atatürk University. Liver samples were taken from each trout. The livers were washed three times with 50 mM Tris-HCl + 0.1 M Na₂SO₄ (pH 8.0) and homogenized by liquid nitrogen (approximately −163°C), transferred to the same buffer, and centrifuged at 4°C, 15,000 × g for 60 min. Supernatant was used in further studies.

Ammonium sulphate precipitation and dialysis

The homogenate was subjected to ammonium sulphate precipitation. For this aim, the homogenate was adjusted to 30–70% saline saturation with solid (NH₄)₂SO₄, respectively. The precipitate was dissolved in a minimum amount of Tris-HCl buffer (50 mM; pH = 8.0). Then, it was dialyzed at 4°C in 1 mM EDTA + 10 mM Tris-HCl buffer (pH 8.0) for 2 h. Partially purified enzyme solution was kept at 4°C.

2′, 5′-ADP Sepharose-4B affinity chromatography

Two grams of dried 2′, 5′-ADP Sepharose-4B was used for a column (1 × 10 cm) of 10 mL bed volume. The gel was washed with 300 mL of distilled water to remove foreign bodies and air, suspended in 0.1 M K-acetate + 0.1 M K-phosphate buffer (pH 6.0), and packed in the column. The column was equilibrated with 50 mM K-phosphate buffer including 1 mM EDTA (pH 6.0). The flow rate was adjusted to 20 mL/h for washing and equilibration. Previously obtained dialyzed sample was loaded onto the 2′, 5′-ADP Sepharose-4B affinity column and the column was washed with 25 mL of 0.1 M K-acetate + 0.1 M K-phosphate, pH 6.0 and with 25 mL of 0.1 M K-acetate + 0.1 M K-phosphate, pH 7.85. Washing was continued with 50 mM K-phosphate buffer including 1 mM EDTA, pH 7.5, until the final difference in the absorbance reached 0.05 at 280 nm. The enzyme was eluted with a gradient of 0 to 0.5 mM GSH + 1 mM NADPH in 50 mM K-phosphate, containing 1 mM EDTA (pH 7.5). Active fractions were collected and dialyzed with equilibration buffer. All procedures were performed at 4°C.

Sephadex G-200 gel filtration chromatography

Dry Sephadex G-200 (3 g) was used for a 165 mL column (2 × 50 cm) bed volume. The gel was incubated in distilled water at 90°C for 5 h and loaded onto the column after removing the air. Flow rate was adjusted to 15 mL/h by means of a peristaltic pump. Then the column was equilibrated with 50 mM Tris-HCl + 50 mM KCl buffer, pH 8.0 until final absorbance difference reached zero at 280 nm. Dialyzed sample was mixed with 5% glycerol. Final sample was loaded onto the column and elutions were collected in 2 mL amounts. In each fraction, enzyme activity was determined at 340 nm. Active fractions were lyophilized and stored at −85°C in order to use in kinetic and electrophoretic studies.

Protein determination

The protein content was quantified spectrophotometrically at 595 nm according to Bradford’s method²¹ for all samples using bovine serum albumin as the standard.

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

The control of enzyme purity, using Laemmli’s procedure²², was carried out in 8% and 3% acrylamide concentrations for running and stacking gel, respectively. Ten percent SDS was added to the gel solution. The gel was stabilized in a solution containing 50% propanol +10% TCA +40% distilled water for 30 min. Staining was done for about 2 h in a solution of 0.1% Coommassie Brillant Blue R-250 + 50% methanol +10% acetic acid. Finally, washing was carried out in the solution of 50% methanol +10% acetic acid +40% distilled water until protein bands were cleared.

In vitro effects of metal ions

In order to determine the effects of the metal ions on fish liver GR, several concentrations of (0.02–1 mM) the metal ions were added into the reaction medium. The enzyme activity was measured and an experiment in the absence of metal ion was used as control (100% activity). IC₅₀ values were obtained from activity (%)–metal ion concentration plots (Figure 1).

Figure 1.  Activity %-[Co⁺²] regression analysis graphs for fish liver GR in the presence of five different cobalt concentrations.

To determine K_i constants in the media with inhibitor, the substrate (GSSG) concentrations were 0.015, 0.04, 0.07, 0.10, and 0.15 mM. Inhibitor (metal ions) solutions were added to the reaction medium, 3 different inhibitor concentrations were used in 1 mL of total reaction volume. Lineweaver-Burk graphs²³ were drawn by using 1/V vs. 1/[S] values, and K_i constant were calculated from these graphs (Figure 2). Regression analysis graphs were drawn for IC₅₀ using inhibition % values by a statistical package (SPSS for windows, version 10.0) on a computer (Student’s t-test; n = 3).

Figure 2.  Line weaver-Burk graph in five different substrate (GSSG) concentrations and in three different cobalt concentrations for determination of K_i..

Results and discussion

In this study, rainbow trout (Oncorhynchus mykiss) liver GR enzyme was purified using simple chromatographic methods. Purification procedure was carried out by the preparation of the homogenate, ammonium sulphate precipitation, and affinity chromatography on 2′, 5′-ADP Sepharose-4B, and gel filtration chromatography on Sephadex G-200. Pyridine nucleotides are able to bind not only glutathione reductase but also to several other proteins.²⁴ For that reason, after 2′, 5′-ADP Sepharose-4B affinity chromatography, Sephadex G-200 gel filtration chromatography was performed. As a result of the consecutive steps, the enzyme was purified up to 1419-fold with a recovery ratio of 38.41% compared to the homogenate (Table 1). After preparation of the homogenate, precipitate saturation of the enzyme was determined as 30–70% with solid (NH₄)₂SO₄.

Table 1.  Purification scheme of GR from rainbow trout (Oncorhynchus mykiss) liver.

Purification step	Total volume (mL)	Activity(U/mL)	Total activity(U)	Protein(mg/mL)	Specific activity(U/mg)	Yield (%)	Purification factor
Homogenate	35	0.243	8.505	15.12	0.0161	100	1
Ammonium sulphate precipitation (30–70%)	18	0.268	4.824	2.41	0.111	56.72	6.89
2′, 5′-ADP Sepharose-4B affinity chromatography	13	0.318	4.134	0.024	13.250	48.61	822.98
Sephadex G-200 gel filtration chromatography	11	0.297	3.267	0.013	22.846	38.41	1419

Besides, Co⁺², Zn⁺², Ca⁺², Fe⁺², Mn⁺², Cr⁺³, Sn⁺² and Mg⁺² were chosen to investigate their inhibitory effects on fish liver GR in this study. Heavy metals have various toxicological effects on living organisms. For example, it has been reported that heavy metals such as mercury and cadmium exist toxic action in a synergistic fashion with salinity^16,25,26. To the best of our knowledge, although the toxic effects of the metal ions have been described, their effects on rainbow trout liver GR have not been studied yet. K_i constants and IC₅₀ values are the most suitable parameters for observing inhibitory effects. As shown in Table 2, IC₅₀ values ranged from 42.2 to 657 µM showing that Co⁺² was the most potent inhibitor although Zn⁺² showed very similar action. However, other metals were effective in low millimolar concentrations. Ki constants for the metal ions ranged from 24.2 to 567 µM in accordance with the IC₅₀ values. Co⁺², Zn⁺², Fe⁺² and Cr⁺³ exhibited competitive inhibition whereas others inhibited the enzyme in non-competitive manner. Similar results were obtained in both in vitro and in vivo studies for various enzymes, such as human carbonic anhydrase isozymes (CA-I and CA-II), human erythrocyte GR, and following enzymes of sparus aurata fish; liver GR, blood catalase, liver catalase, glutathione peroxidase^16,25,27. Contrarily, Mg⁺² showed activatory effect in the concentration range of 0.05–5 mM.

Table 2.  K_i and IC₅₀ values obtained from regression analysis graphs for fish liver GR in the presence of different metal ion concentrations.

Metal ions	IC50 (mM)	Ki (mM)	Inhibition type
Co^+2	0.0422	0.0242 ± 0.001	Competitive
Zn^+2	0.0631	0.0598 ± 0.003	Competitive
Ca^+2	0.3570	0.298 ± 0.012	Non-competitive
Fe^+2	0.486	0.364 ± 0.011	Competitive
Mn^+2	0.508	0.398 ± 0.013	Non-competitive
Cr^+3	0.592	0.441 ± 0.018	Competitive
Sn^+2	0.657	0.567 ± 0.009	Non-competitive
Cd^+2*	0.0655	0.104 ± 0.001	Competitive
Cu^+2*	0.0820	0.117 ± 0.001	Competitive
Pb^+2*	0.1220	-	-
Hg^+2*	0.509	-	-
Fe^+3*	0.797	-	-
Al^+3*	0.804	-	-
Mg^+2	A	-	-

*Represents the data from reference 2.

A, activator for 0.05–5 mM.

Studies regarding undesirable effects of cobalt, zinc or calcium ions on various components of the metabolism have gained particular attention over the recent years. For instance, Ekinci et al (2011) demonstrated that micromolar cobalt and zinc exposure attenuates the expression of insulin-like growth factors and growth hormone in rainbow trout. The study proposed that IGF/GH axis may be affected by micromolar exposure to these metals²⁸.

Nemery et al (2003) carried out several experiments in which they measured indices of oxidant stress, mainly changes in the oxidation state of glutathione and in the activity of the pentose phosphate pathway, upon exposure of hamster pulmonary tissue to CoCl₂ in vivo by intra-tracheal instillation or in vitro by incubating lung slices. Their experiments indicated that cobalt ions are capable of causing thiol oxidation in lung tissue as an early manifestation of oxidant stress, but more studies are needed to establish the relevance of this mechanism in the causation of lung disease in subjects exposed to cobalt-containing dusts.²⁹

There are many studies concerning the effects of environmental conditions on the activity of antioxidant enzymes and oxidative stress. For instance, two common rubber additives, 2-mercaptobenzothiazole (MBT) and diphenylamine (DPA) were reported to cause an increase in hepatic GR activity as well indicating that GR is very susceptible to environmental conditions.³⁰ Additionally, increasing stocking density was demonstrated to inhibit the antioxidant enzyme GR in rainbow trout recently.³¹ Cobalt and zinc exposure of rainbow trout was also showed to increase the expression of metallothioneins and cytocrome P450.³²

In this investigation, metal ions showed high inhibitory effects on rainbow trout liver GR enzyme activity. Nowadays, the number of industry wastes and metal refuses are rising in rivers, lakes and seas in the world. Thus, undesirable side effects of these metal ions on GR activity, body metabolism and GSH synthesis can be reduced for fish and fish eaters by concerning these obtained K_i and IC₅₀ values.

Declaration of interest

The authors report no conflicts of interest.